Critical Role Of AAF1 Gene In C. Albicans Biofilm Formation In Vitro And In Vivo

Objectives: Infection rates caused by Candida albicans have been incraesingwith exceptionally high mortality. Biofilm formation is a major virulence factor inthe pathogenicity of Candida albcans, and they are difficult to eradicate especiallybecause of their very high antifungal resistance. Although there were some studieshave shed some light, molecular mechanisms that govern biofilm formation andpathogenicity still await full clarification. In this paper the role of AAF1 in biofilmformation will be elplored.Methods: Firstly, standard in vitro gene splicing techniques were utilized toconstruct plasmid pHS1-U3du3-HS2 uesd in gene disruption strategy asmini-Ura-blaster cassette and plasmid pHS1-GAAU3-HS2 used to overexpressAafl. Using mini-Ura-blaster technology, theâ–³aafl/â–³aafl homozygous deletionmutant strain M2-2(â–³aafl/â–³aafl) was made. Then the strainM3(â–³aafl/â–³aafl+pGAP-AAF1) overexpressing Aafl was obtained bytransformed M2-2 with plasmid pHS1-GAAU3-HS2. All Strains were confirmedby PCR using different primers. And Western blot was applied to detect Aaflprotein. Biofilm formation in vitro and in vito by strain CAI4, M2-2 and M3 wereobserved by invert microscope and scanning electron microscope. The reversetranscription and quantitative polymerase chain reaction (qRT-PCR) analysis wasused to compare mRNA abundances of the HWP1 gene and ALS3 gene betweenstrain M3 and strain M2-2 at 48h ofbiofilm development in vitro.Results: Plasmid pHS1-U3du3-HS2 and plasmid pHS1-GAAU3-HS2constructions were made successivly. The AAF1 gene was disrupted completely andâ–³aafl/â–³aafl deletion mutant strain M2-2 was made. The complemented strain M3(containing AAF1 ORF) was constructed by transforming M2-2 (â–³aafl/â–³aafl)with plasmid pHS1-GAAU3-HS2. And the Aafl protein was confirmed by western blot. The microscope imaging revealed thatâ–³aafl/â–³aafl mutant (strain M2-2)displayed a severe defect in biofilm formation compared to the reference Stain(CAI4), and introduction of a single wild-type AAF1 allele (strain M3) rescued thedefect substantially in vitro biofilm formation model. Biofilm formatiom wasvisualized after 48h by scanning electron microscopy of the intealuminal cathetersurface taken from a rat venous. The wild-type CAI4 andâ–³aafl/â–³aafl+pGAP-AAF1 complemented strains M3 yielded extensive adherent populations andabundant matrix material. In constrast, theâ–³aafl/â–³aafl mutant yielded fewadherent cells and was devoid of biofilmmaterial. At 48h of biofilm development invitro,Total RNA was extrated and was measure by qRT-PCR analysis. The foldincrease in mRNA for ALS3 was 15.63492 inâ–³aafl/â–³aafl+pGAP-AAF1complemented strains M3 as compared withâ–³aafl/â–³aafl mutant strains M2-2.HWP1 was expressed at 8.817685-fold-higher levels in the complemented strainsM3 (â–³aafl/â–³aafl+pGAP-AAF1)than in theâ–³aafl/â–³aafl mutant strains M2-2.Conclusions: The constructions of plasmid pHS1-U3du3-HS2,pHS1-GAAU3-HS2 and strainsâ–³aafl/â–³aafl mutant,â–³aafl/â–³aafl+pGAP-AAF1 complemented were made correctly.This study demonstrates that therole of AAF1 was critical in Candida albicans biofilm formation in vitro and in vito.And the mechamism may be Als3-mediated and Hwpl-mediated adhered.